Two selenocysteine tRNA isoacceptors which function to read the termination codon, UGA, in protein synthesis (and thus donate selenocysteine to the growing polypeptide chain in response to specific UGA codons) and to serve as carrier molecules for the biosynthesis of selenocysteine have been identified in mammalian cells. The pathway for selenocysteine biosynthesis proceeds from tRNA[Ser]Sec ---> seryl- tRNA[Ser]Sec ---> phosphoseryl-tRNA[Ser]Sec ---> selenocysteyl-tRNA[Ser]- Sec. Both isoacceptors were sequenced from rat liver and differ from each other by a single base in the wobble position of the anticodon. One isoacceptor, designated CmCA on the basis of its anticodon sequence, contains 0-methylcytidine and the other, designated NCA, contains an unidentified (presumably a modified uridine) base in this position. Since both tRNAs arise from a single copy gene (where the gene contains a thymidine at the wobble position), one of the isoacceptors must be an edited species. The pathway of synthesis of the tRNAs has been reconstituted in Xenopus oocytes following microinjection of the gene or of the RNA (generated from the human gene cloned into an expression vector). The pathway proceeds from the tRNA[Ser]Sec gene ---> processed primary transcript ---> NCA ---> CmCA. As observed in mammalian cells in culture and in specific rat tissues, the tRNA[Ser]Sec population in oocytes is responsive to selenium. The changes in primary structure at each biosynthetic step are presently being characterized by the process of polymerase chain reaction, and by fingerprint and minor base analysis. A selenocysteine tRNA that decodes UGA has been identified in animals, plants, fungi and two very divergent protists; the universal genetic code has therefore been expanded to include selenocysteine as the 21st naturally occurring amino acid.